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First developed and most widely used technology for generation of monclonal antibodies = the use of mouse hybridomas. It is generated from stable fusion of immortalised myeloma cells with B cells from immunnized mice.

Method:

mice first exposed to antigen(Ag) to which AB of interest is specific for –> by series of injections of Ag

B cells then isolated from spleen

myeloma cells are aslo selected to ensure that they themselves do not secrete AB and lack hypoxanthine guanin phosphoribosyltransferase (HGPRT) gene , making them sensitive to HAT medium

aminopterin blocks nucleotide synthesis. Therefore cells forced to use the purine salvage pathway to synthesise purine nucleotide which require HGPRT , as B cells have this gene , so only B cells that have fused will be able to survive.

unfused myeloma cells is removed for its potential to outgrow other cells

B-cell myeloma hybrids produce AB that are immortal

the incubated medium is diluted into multi well plate to extent that each well contains one cell

so AB produced in single cell , were generated from single B cells = directed towards same epitope and thus monoclonal AB

Rituximab is an example of a chimeric mAb that is used clinically to treat non-Hodgkins lymphoma . Unfortunately, even the murine variable region can still be recognised as foreign—leading in many cases to the same limitations as murine mAbs.

The simplest of many humanization strategies involves transfer of complementarity determining regions (CDR) from mouse AB to human IgG. For generation of high affinity binding , generally requires additional transfer of one or more framework-region residues from parent mouse AB.

Combinatorial library(fewer than 107 scFv genes) was then cloned for display on surface of phage,

then used to identify scFv that bind target antigens.

More commonly,antibodies need to be optimized to meet the design goals of the project, and this is readily accomplished by selection for high-affinity variants from phage-display
libraries.

One human antibody isolated by phage-display technology has been approved widely for therapeutic use: adalimumab ( TNF- specific monoclonal AB) used for treatment of rheumatoid and psoriatic arthritis.

A particular strength of phage-display libraries, in contrast to hybridoma technology, is that direct
selection for exquisitely specific binding properties, such as species crossreactivity, is sometimes possible( allowing the biological function of the antibodies to be evaluated in animal models of disease.)

Antibody(AB) is useful tool for research and medicine ( diagnosis & therapy). In fact it emerged in the mid 1990’s as a new drug class . AB was approved for therapeutic use in clinical setting such as

oncology;

chronic inflammatory diseases;

transplantation;

infectious diseases and

cardiovascular medicine

One of the biggest advantage of AB use is the high success rate from first use in humans.

29% chimeric AB

25% humanized AB

this is compares favorably with 11% success rate of small molecule drugs

In general AB are well tolerated by humans although infusion reactions( fever, chills e.g patients treated with rituximab CD20 specific monoclonal AB) particularly for first dose are common but manageable. And key strength of AB as therapeutics is their clinical potential can be readily be increased by improving their existing properties.

AB limitations of therapeutics

restriction of targets to those on surface or exterior of host cells or invading pathogens

AB drugs are expensive which limits its use to serious medical conditions. Many factors contribute to high cost a) large expense of drug development in general, b) high cost of manufacturing and c) large total doses that are often required.

although AB therapeutics often safe and well tolerated, rare but serious adverse events have been reported.

Goal for AB therapeutics

One need is to improve the efficacyof AB therapeutics e.g anticancer therapy, for which AB are seldom( if ever) curative. Therefore in oncology, one of the goals of increasing efficacy of AB is to improve, antitumour activity and improve patient survival.

How? by increasing frequency of partial responses or more preferably , complete responses and by extending the duration of responses.

To improve safety, might be useful to focus on limiting first infusion reactions or more serious target related adverse events

e.g acute and severe influenza- like syndrome associated with adminstration of CD3 specific monoclonal AB= due to Fc-mediated and can be overcome…

how? by attenuating the interaction between Fc region of AB and receptors for AB(e.g IgG receptors: FcγRs) expressed by patient

Increasing the potency of AB or extending its half-life in plasma might allow dose or frequency of administration to be reduced, which might have associated benefits e.g improved quality of life and/or convenience for patient and or reduced cost of drug.